A wide variety of naturally expressed anti-microbial peptides (AMPs) have been discovered in plants, animals, and humans. AMPs are remarkably for their general ability to halt growth of both Gram negative and Gram positive bacteria. Cationic AMPs are known to form amphipathic helices that bind to anionic cell membranes, form pores, and eventually lyse the cell. The relative inability of bacteria to resist this general mode of action makes AMPs and their mimics interesting drug candidates against antibiotic-resistant strains. While it is well established that AMPs degrade bacterial membranes and eventually lead to lysis, most of the mechanistic work has focused either on studies of synthetic vesicles in vitro or on long-time, bulk effects of AMPs on bacteria. The detailed mechanism of AMP attack on bacterial membranes is not well understood. The primary goal of this work is to develop novel fluorescence microscopy assays that directly observe the attack of AMPs on bacterial membranes in real time, for individual cells. For the first time, these methods will enable placement of a number of directly observable events on the same, unified time line covering the first 10 seconds to the first two hours after injection of the AMP. Measured events will include AMP binding density, halting of flagellar motion, cessation of growth, leakage of the periplasm, and leakage of the cytoplasm. Time lapse imaging after removal of the AMP will reveal which symptoms of the attack are reversible and which are not, as well as the time scale on which cells recover growth. The new methods will be used in an exploratory study of the mechanisms by which a variety of different AMPs attack membranes in both the Gram negative E. coli and the Gram positive Bacillus subtilis. These include LL-37, cecropin A, magainin-2, melittin, alamethicin, indolicidin, bactenecin-5, 1-defensin, and protegrin-1. We will directly compare the surface concentrations at which each AMP degrades bacterial cell membranes with results of previous studies in vitro on synthetic lipid bilayers. The results will help determine whether purported carpet- forming and pore-forming mechanisms gleaned from in vitro studies are relevant to real bacterial membranes. The methods developed here will be widely applicable to studies of both AMPs and drug candidates interacting with a variety of bacterial strains. PUBLIC HEALTH RELEVANCE: Bacteria are increasingly resistant to drugs. Antimicrobial peptides are the front-line defense against pathogens throughout the animal kingdom, but we do not really understand how they work to kill bacterial cells. This work will develop novel imaging methods that will enable direct observation of the mechanisms of bacterial killing by antimicrobials with better spatial and time resolution than ever before.